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Re: Molecular Biology Collection Thread

Transcription Initiation in Bacteria

It's a complicated process, including 1) formation of a closed promoter complex; 2) conversion of the closed promoter complex to an open promoter complex; 3) polymerizing the first few nucleotides (up to 10) while the polymerase remains at the promoter, in an initial transcribing complex; 4) promoter clearance, in which the transcript becomes long enough to form a stable hybrid with the template strand. This helps to stabilize the transcription complex, and the polymerase changes to its elongation conformation and moves away from the promoter.

Re: Molecular Biology Collection Thread

Tips:

*.The key player in the transcription process is RNA polymerase. The E.coli enzyme is composed of a core, which contains the basic transcription machinery, and a sigma-factor, which directs the core to transcribe specific gene.
The separation of core enzyme and sigma-factor causes a profound change in the enzyme's activity. Whereas the holoenzyme could transcribe intact phage T4 DNA in vitro quite actively, the core enzyme has little ability to do this. On the other hand, core polymerase retains its basic RNA polymerizing function because it could still transcribe highly nicked templates (DNAs with single-stranded breaks) very well.

Sigma as a specificity factor
Adding sigma back to the core reconstituted the enzyme's ability to transcribe unnicked T4 DNA. Even more significantly, the holoenzyme transcribed only a certain class of T4 genes, but the core showed no such specificity. Also, core enzyme transcribes both DNA strands, which is unnatural.

*.The sigma-factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter. This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by sigma. The sigma-factor can therefore select which genes will be transcribed.The polymerase holoenzyme binds much more tightly to the T7 DNA than does the core enzyme. In fact, the holoenzyme dissociates with a half life time of 30-60 h, which lies far beyond the timescale. This means that after 30-60 h, only half of the complex had dissociated, which indicates very tight binding indeed. By contrast, the core polymerase dissociated with a half life time of less than a minute, so it bound much less tightly than the holoenzyme did. Thus, the sigma-factor can promote tight binding, at least to certain DNA sites.

*.Sigma stimulates initiation, but not elongation, of transcription.

*.At some point after sigma has participated in initiation, it appears to dissociate from the core polymerase, leaving the core to carry out elongation. Furthermore, sigma can be reused by different core polymerases.

*.The sigma-factor appears to be released from the core polymerase, but not usually immediately upon promoter clearance. Rather, sigma seems to exit from the elongation complex in a stochastic manner during the elongation process.

*.On binding to a promoter, RNA polymerase causes melting that has been estimated at 10-17 bp in the vicinity of the transcription start site. This transcription bubble moves with the polymerase, exposing the template strand so it can be transcribed.